Abstract

Bacterial infections of the dental pulp result in soft tissue and alveolar bone destruction. It has been suggested that Th1 responses promote disease, whereas Th2 responses are protective. However, other studies have challenged this notion. To address this question, bone destruction was evaluated in mice immunized to develop strong and polarized Th1- or Th2-biased responses to the oral pathogen Porphyromonas gingivalis. Th1 bias was confirmed by the presence of high titers of serum IgG2a and the production of high levels of interferon (IFN)-gamma and no interleukin (IL)-4 by lymph node cells stimulated with P. gingivalis antigens. In contrast, Th2-biased animals had high titer IgG1 and no IgG2a, and their lymph node cells produced high levels of IL-4 but no IFN-gamma. Subsequent infection of the dental pulp with P. gingivalis caused extensive inflammation and alveolar bone destruction in Th1-biased mice, whereas Th2-biased mice and controls developed minimal lesions. Inflammatory granulomas in Th1-biased mice were heavily infiltrated with osteoclasts and had high local expression of IFN-gamma, IL-1alpha, and IL-1beta. Little or no IFN-gamma/IL-1alpha/IL-1beta and no obvious osteoclasts were detected in lesions of Th2-biased and control groups. These results directly demonstrate that specific Th1 responses promote severe infection-stimulated alveolar bone loss.

Local inflammation in mice immunized with P. gingivalis antigens under Th1 or Th2 conditions. C57BL/6 mice (three per group) were immunized in the rear footpads with 10 μg of P. gingivalis a soluble lysate antigens (Pg) preparation mixed with alum (25 μg) (Th2) or with alum plus the cytokine IL-12 (1 μg) (Th1). Mice were boosted 4 weeks later using the same antigen/adjuvant formulations used for primary sensitization. Massive swelling was observed in the footpads of mice immunized with the Th1 versus Th2 formulation (A). A quantitative measurement of the footpad swelling is depicted in B. After immunization, mice were sacrificed, and their feet were amputated and decalcified. C: Sections (5 μm) were obtained from the inflammation site and H&E-stained. Note that the inflammatory cells are predominantly mononuclear and are noticeably more abundant in the Pg plus alum plus IL-12 immunized mice compared with the mice immunized with Pg plus alum.

Isotype-specific antibody response of Th1- and Th2-biased mice. Anti-P. gingivalis antibody responses of IgG1 (A) and IgG2a (B) isotypes were measured by ELISA. Sera were obtained from the immunized mice described in Figure 1.

Antigen-induced proliferation of lymph node cells from Th1- and Th2-biased mice. C57BL/6 mice (three per group) were immunized as described in Figure 1. One week after boost, mice were sacrificed, and lymphocytes were obtained from popliteal lymph nodes and cultured for 3 days in the presence of medium containing P. gingivalis lysate antigens. Proliferation was measured by incorporation of [3H]thymidine added during the last 12 hours of culture. Results are expressed as the mean ± SD of triplicate cultures.

Production of cytokines by lymph node cells of Th1- and Th2-biased mice. Culture supernatants harvested at the end of the cultures described in Figure 3 were assayed for the presence of IFN-γ (A) and IL-4 (B) by sandwich ELISA. Bars are the SD of triplicate cultures.

Periapical bone resorption caused by P. gingivalis in Th1- and Th2-biased mice. C57BL/6 mice (10 per group) were immunized as described in Figure 1. Four weeks after the second immunization, the mice were subjected to intrapulpal infection with P. gingivalis. Alveolar bone resorption was analyzed in the periapical region of the mandibular first molars by microcomputed tomography after 21 days. Representative images are shown. Note the extensive bone resorption (arrow) in Th1-biased mice compared with all other groups. The areas of periapical bone loss per group of mice were calculated from the microcomputed tomography images and are expressed in square millimeters. Bars represent the mean ± SD (n = 10/group).

Histology of periapical tissues of Th1- and Th2-biased mice. Tissues obtained from mice immunized and infected as described in Figure 5 were decalcified and H&E-stained. Note the massive mononuclear cell infiltrate in nonorganized granuloma lesions present in Th1-biased animals. Arrows indicate intense infiltration of osteoclasts in lesions of Th1-biased mice. Th2-biased mice had a noticeably less abundant cellular infiltrate and no obvious presence of osteoclasts.

Local expression of cytokines in the periapical granulomas of Th1- and Th2-biased mice. Proteins were extracted from periapical lesion tissues from the mice described in Figure 5 and were tested for the presence of cytokines by sandwich ELISA. Results are expressed as picograms of cytokine per milligram of periapical tissue and represent the mean ± SD of the measurements obtained from 10 mice per group. Neither IL-4 nor IL-10 was detected in any sample (not shown).

Immunohistochemical staining for IFN-γ and IL-1α/β in periapical granulomas. Periapical tissues obtained from mice immunized and infected as described in Figure 5 were decalcified, formalin-fixed, and embedded in paraffin. Sections of 5-μm thickness were obtained and processed as described in Materials and Methods. Note the abundant expression of both IFN-γ and IL-1α/β (brown and dark brown colors) in Th1-biased mice. R, distal root, first mandibular molar; B, alveolar bone; P, periapical granuloma.